This invention is in the general field of pharmaceuticals for treatment of conditions caused by xenoreactive antibodies.
Organ Transplantation
Organ transplantation is now widely viewed as the preferred treatment for end stage organ failure, based on both a quality of life and cost basis. One year graft survival for kidney and heart transplantation at the major transplant centers is now greater than 90% with acute rejection rates less than 30%. There are approximately 2500 heart transplants and 12,500 kidney transplants performed every year in the USA. The major limitation to more widespread utilization of this procedure is a shortage of available organs. This can be seen by looking at the transplant waiting list, which shows that more than twice the number of people are waiting for an organ than could ever receive one. Additionally, 10 people per day die while waiting for a transplant. The demand for organs, however, is much greater than the number of people on the transplant waiting list. There are 125,000 patients in end stage renal failure who theoretically could benefit from a transplant. It is also been estimated that 25,000 to 40,000 people could benefit from a heart transplant if a donor organ were available.
The availability of human organs is not likely to increase in any significant way over the next few years and even if all of the potential human donor organs could be utilized this would only increase the number of organs transplanted by about 2 fold, far less than the actual demand. Possible mechanical solutions to this problem such as left ventricular assist devices for cardiac failure while adequate at least for temporary support appear unlikely to provide for a long-term solution and as yet no implantable device exists for a kidney. Indeed, renal transplantation is viewed as superior and preferred in terms of the quality of life compared to dialysis. Given these alternatives the medical and scientific communities have turned to animals to provide for a solution to the organ shortage problem.
Animals as Donors
The preferred animal species for transplantation to humans is the pig. Although the closely related non-human primate species are immunologically more similar to humans, and therefore the rejection process which is seen upon transplantation could likely be more easily controlled by current immunosuppressive drugs, a number of reasons make this possibility remote. First, a non-human primate of appropriate size for a heart transplant into an adult human would be a chimpanzee or equivalent large animal. These animals are on the endangered species list and thus would raise significant ethical questions even if it were possible to breed large numbers in captivity. The most abundant non-human primate, which is the baboon, is small and could not provide hearts for transplantation into adult humans although conceivably kidneys or livers would be possible. However, the baboon contains a number of potentially pathogenic organisms which could present a problem if transmitted to a human, and the generation of large numbers of specific pathogen free animals would be extremely costly and time consuming. The pig, a domesticated farm animal, on the other hand is of an appropriate size, can be raised to obtain specific pathogen free animals and is available in large numbers. Clearly, if the immunological problems could be overcome the pig would be the ideal donor to solve the organ shortage problem.
The Immunological Problem
The initial barrier to transplantation of a pig organ to primates is a process of hyperacute rejection which results in the loss of the graft within a few minutes to hours of transplantation and is characterized histologically by thrombosis, hemorrhage, edema and a lack of a cellular infiltrate (Platt, J. L., et al., Immunology Today 11:450-456, 1990). This process is initiated by the binding of antibody, which is already present in the recipient, to the graft endothelium, and the resulting activation of the complement cascade. A similar process can be seen in allografts when antibody is present in the recipient prior to transplantation. In thinking about the pig as a donor, it is useful to review the effect of pre-existing antibodies on allograft survival and what strategies are important in obtaining prolonged graft survival.
The presence of antibody in the serum of an allograft recipient, which recognizes antigen present on the donor endothelium, can be an indicator of a poor prognosis for long-term graft survival. Indeed, in a percentage of these allografts a rapid process of rejection, as described above for pig to primate xenografts, in which the graft is lost within a few minutes to hours after reperfusion, can occur. An example of this situation is seen when the donor and recipient are incompatible with regard to the blood group ABO system. The blood group antigen, which is a carbohydrate, is present on the donor endothelium. When a graft from a blood group A donor is transplanted into a blood group O recipient, in which antibody against the blood group A antigen is present, antibody is deposited in the graft. In the case of these grafts in which hyperacute rejection occurs the binding of antibody to the graft endothelium causes complement to be fully activated which results in damage to the endothelium and subsequent loss of the endothelial barrier function resulting in thrombosis, hemorrhage, edema and irreversible graft failure. If hyperacute rejection does not occur many, though clearly not all, of the grafts are lost due to vascular rejection in the subsequent weeks to months. These transplants were initially performed in the pre-cyclosporin era and led to the suggestion that matching for ABO compatibility should be performed prior to transplantation.
As the success of allografts improved, dramatic shortages in the availability of organs led a number of groups to reassess the possibility of transplanting across the ABO barrier particularly in the case of living related kidney donors. These investigators found that in order to routinely prevent hyperacute rejection and achieve optimal long term graft survival, comparable to that obtained with ABO matched grafts, the antibody present at the time of transplant has to be temporarily removed usually by physical means such as immunoapheresis. A standard or in some cases an enhanced immunosuppression protocol is then applied. Under these circumstances the long-term outcome for these grafts is as good as would be obtained with ABO compatible grafts. In a percentage of these ABO incompatible grafts antibody apparently returns to the circulation but the graft is not rejected and this phenomenon has been termed xe2x80x9caccommodationxe2x80x9d. However, it is still far from clear that this antibody as detected in standard haemagglutination reactions is actually capable of binding to the graft. In many circumstances the antibody does not return in a detectable form to the circulation, although it is certainly conceivable that antibody is bound to the graft.
In a pig to primate xenograft, the xenoreactive antibody which is found in the recipient prior to transplant predominantly if not exclusively recognizes the unique carbohydrate structure, xcex1Gal(1,3)Gal. This structure is found as the terminal sugar on glycolipids and glycoproteins present on the donor pig endothelium. The pig and many other mammals but not humans or Old World monkeys synthesize this epitope. This scenario is in many ways reminiscent of the ABO blood group mismatched allografts described above. In a similar fashion to the mammalian blood group antigens such as the human blood group A, the lack of the antigen in an animal results in the synthesis of antibodies which recognize it. This antibody is probably induced and stimulated on an ongoing basis by gut bacteria, which possess a related structure. Indeed, Galili et. el (1988) showed that anti-Gal antibodies bound to a variety of enterobacteria such as E.coli, klebsiella and salmonella. The Gal reactive structure varied between the bacteria and was either the lipopolysaccharide or the capsular polysaccharide (Hamadeh et. el. 1992). Both of these classes of antigens are well known as T-cell independent antigens. Additionally, the isotype of the ABO and xcex1-Gal antibodies are similar, the levels of antibody in the serum are similar and the density of the carbohydrate antigens on the donor endothelium are also within the same range. However, when a pig to primate organ transplant is performed the graft is almost always rejected within a few minutes to hours with the very occasional graft lasting at most several days. This is in direct contrast with what happens in the allograft where the occasional graft is hyperacutely rejected and the majority survive for prolonged periods of time (weeks to months). A significant difference between the survival of allografts and xenografts under these conditions is most likely due to the incompatibility of the complement regulatory proteins. In the xenografts, antibody binds to the graft endothelium and results in the activation of the complement cascade. In the case of the allograft there are a series of proteins present on the donor endothelium which regulate the activity of complement and can be thought of as a primitive self/non-self-recognition process whereby if complement is inadvertently activated on the endothelium it can be inactivated before damage occurs. These proteins include Decay Accelerating Factor (DAF: CD55), Membrane Cofactor Protein (MCP: CD46) and CD59. In the case of the pig, the pig equivalent of the human CRP""s does not function effectively to limit the action of human complement. Therefore, when complement is activated in the xenograft due to the functional lack of the CRP""s the endothelium is severely damaged resulting in thrombosis, hemorrhage, edema and graft loss. This hypothesis has been confirmed by the generation of transgenic pigs, which express human CRP""s and routinely overcome hyperacute rejection.
If this were the only difference between an allograft and a xenograft then one might assume that with the addition of immunosuppression or antibody removal plus immunosuppression and the use of transgenic animals that one would observe xenograft survival with a similar time course as in an allograft. However, the simple addition of immmunosuppression does not substantially prolong graft survival and the organ invariably succumbs to vascular rejection. Antibody removal at the time of transplant combined with immunosuppression can prolong survival but not reach survival levels comparable with an allograft and again vascular rejection inevitably ensues. This vascular rejection is characterized histologically like hyperacute rejection by hemorrhage, thrombosis, and a lack of a cellular infiltrate; however, unlike hyperacute rejection there are significant regions of ischaemia. Immunopathological analysis reveals that antibody is bound to the graft but very little complement components are deposited.
There are exceptions to this course of events and these are found when exceptionally high doses of immunosuppressive agents, whose mechanism of action is related to their anti-proliferate effect, are utilized or the physical removal of antibody by immunoapheresis in the post transplant period is aggressively continued. Taken together with the lack of a cellular infiltrate these results suggest that the organs are being lost due to a vascular rejection process which is most likely an antibody mediated event although the mechanism whereby antibody causes this rejection process are not yet clear.
When the graft is present in the recipient, it is difficult to determine the level and specificity of xenoreactive antibody because the graft can act as a sink and bind up the antibody. We sought to circumvent this problem by following the antibodies in the animal after the removal of a rejected non life supporting heterotopic heart transplant and at the same time maintain the immunosuppressive regime after the graft is removed. Under these circumstances soon after the graft removal and in the following few days we observe a rapid rise in xenoreactive antibody levels in the animal. This is characterized by a 4-10 fold increase in IgM and a 100-200-fold increase in IgG. These antibodies almost exclusively recognize the xcex1Gal(1,3)Gal structure. A similar rise in xcex1Gal(1,3)Gal antibodies is seen in humans who are the recipients of cellular pig xenografts such as pancreatic islets or who have been exposed to a pig liver in an ex vivo perfusion circuit. These data suggest that the grafts are lost due to the enhanced production of xcex1Gal(1,3)Gal antibodies.
The observation that the antibodies which we hypothesize mediate graft rejection in the pig to primate species combination predominantly recognize a single chemically defined epitope raises the possibility to control this antibody production in a specific manner. Specificity in the control of the immune response would be advantageous because it would reduce the risk of infectious complications associated with generalized immunosuppression. As discussed above, these responses are not easily controlled by current immunosuppression at clinically relevant levels making the rationale for a greater specificity even more important.
In vivo neutralization of antibody by the administration of soluble ligand has been proposed for both ABO incompatible allografts and pig to primate xenografts. In both systems the infusion of large amounts of sugar are required due to a combination of low affinity of the ligand for antibody and short half life in the serum. In experimental ABO incompatible baboon to baboon heart allografts, if sugar infusion is maintained for a number of days in the presence of immunosuppressive agents the infusion can be stopped and long term graft survival can be achieved. In many ways this is reminiscent of the observation that the temporary removal of antibody by physical means, for example plasma exchange or specific immunopheresis in combination with immunosuppression, can result in long-term graft survival.
In the case of the pig to primate xenograft the administration of soluble forms of the xcex1Gal(1,3)Gal carbohydrate has prevented hyperacute rejection, however, as soon as the sugar infusion is stopped the graft is rejected. To date the infusion has not been maintained for a prolonged enough period of time to assess when, if at all, the infusion can be stopped, as was the case with the allograft. It is conceivable that there is a substantial difference between allograft and xenograft rejection such that infusion cannot be stopped in the vast majority of cases. This experiment could be at least conceptually improved if it were possible to either dramatically improve the half-life of the sugar or, increase the affinity or both such that much less was required or dosing was less frequent.
One aspect of the present invention provides a polymer, synthesized by a cross-coupling reaction of
(i) polymer scaffold units ,comprising a plurality of nucleophilic or electrophilic functional groups, and
(ii) xenoantigens comprising a functional group capable of reacting with the functional groups presented on the polymer scaffold unit,
wherein the reaction is carried out in the presence of a crosslinking reagent under conditions wherein polymer scaffold units are coupled with xenoantigens, and separate polymer scaffold units are cross-linked to one another.
In certain embodiments, the functional groups for the polymer scaffold units and xenoantigens are selected from the group consisting of amine and carboxylic acid, alcohol and alkyl halide or sulfonate, thiol and alkyl halide or sulfonate, phosphine and alkyl halide or sulfonate, phosphite and alkyl halide or sulfonate, and aldehyde or ketone and amine. In preferred embodiments, the functional groups produce an amide, ether, thioether, or phosphate linkage between the polymer scaffold unit and xenoantigen upon cross-coupling.
The cross-coupling may be carried out, e.g., in the presence of an activating agent that activates the functional groups of the polymer scaffold units and xenoantigens for forming a covalent bond there between. For instance, the activating group may be a dehydrating agents, Bronsted acid, Bronsted base, Lewis acid, Lewis base, acyl halide, or a phosphoryl halide. In certain preferred embodiments, the activating agent is a diimide, e.g., dicyclohexyldiimide [DCC] or ethyl-3-(dimethylamino)propyldiimide [EDC].
In certain embodiments, the polymer scaffold units are pharmacologically-acceptable, non-immunogenic molecules. For instance, the polymer scaffold units can be polyethylene glycol, an oligopeptide, or oligosaccharide (e.g., qextran, dextrin or a cyclodextrin) bearing nucleophilic or electrophilic functional groups. Suitable nucleophilic or electrophilic functional groups include amines, alcohols, thiols, selenols, phosphines, aldehydes, ketones, acid chlorides, acids, esters, alkyl halides, and alkyl sulfonates. In particular embodiments, the functional group on the polymer scaffold unit is an amino or carboxylate moiety, and the functional group of the xenoantigen reacts therewith to form an amide linkage.
In certain preferred embodiments, the polymer scaffold unit is as multiply-aminated polyethylene glycol, e.g., octa(amino)polyethylene glycol. In additional embodiments, the polymer scaffold unit is aminated dextran or an aminated cyclodextrin. In further embodiments, the polymer scaffold is a dendridic polyamine.
The xenoantigen can be, to illustrate, a carbohydrate, a peptide, a glycopeptide or a lipid. In certain preferred embodiments, xenoantigen includes an epitope which is cross-reactive with an xenoreactive antibody against xcex1-galactosyl moieties, and more preferably the xenoantigen is an oligosaccharide, e.g., having an xcex1Gal(1,3)Gal moiety represented by the general formula: 
wherein:
R, independently for each occurrence, represents H or a C1-C6 alkyl or other hydroxyl protecting group;
Rxe2x80x2 is absent or represents an oligosacchride consisting of 1-8 saccharide residues; and
X represents a bond or linker moiety linking the oligosacchride moiety to the polymer backbone, e.g., preferably being a cyclic, branched or straight chain aliphatic group of 2-10 bonds in length, cycloalkyl, alkenyls, cycloalkenyl, alkynyl, aryl, heteroalkyl, or heteroaryl moiety.
For example, the xenoantigen can include an oligosacchride having represented by one of the general formula: 
wherein,
R1, independently for each ocurrence, is H, xe2x80x94OR, xe2x80x94SR or xe2x80x94N(H)xe2x80x94Yxe2x80x94R;
R, independently for each occurrence, represents H or a C1-C6 alkyl or other hydroxyl protecting group;
R2 is a C1-C6 alkyl;
Y, independently for each occurrence, is absent, or xe2x80x94C(S)xe2x80x94, xe2x80x94S(O)2xe2x80x94, xe2x80x94C(O)xe2x80x94Axe2x80x94;
X represents a bond or linker moiety linking the oligosacchride moiety to the polymer backbone;
Z is O or S; and
A is xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94(C1-C6 alkyl)-, xe2x80x94NHxe2x80x94(C1-C6 alkenyl)-, xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94(C1-C6 alkyl)-, xe2x80x94Oxe2x80x94(C1-C6 alkenyl)-, xe2x80x94Sxe2x80x94, xe2x80x94Sxe2x80x94(C1-C6 alkyl)-, xe2x80x94Sxe2x80x94(C1-C6 alkenyl)-.
In certain preferred embodiments, the xenoantigen includes one or more of: xcex1Gal(1,3) xcex2Gal; xcex1Gal(1,3)xcex2Gal(1,4)xcex2GlcNAc; or xcex1Gal(1,3)xcex2Gal(1,4)xcex2Glc moieties. In other embodiments, the xenoantigen may include one or more of: xcex1Gal(1,2)Gal, xcex1Gal(1,4)Gal, xcex2Gal(1,3)GalNAc or 3-O-sulphated galactose (SO4-3Gal).
The xenoantigen can be a di-, tri-, tetra- and penta-oligosacchrides.
In certain embodiments, the polymer is homogenous with respect to the xenoantigen displayed thereon. In other embodiments, the polymer includes two or more different xenoantigens, e.g., on the same molecule.
In particular preferred embodiments, the crosslinking agent has at least two functional groups capable of reacting with the polymer scaffold units, the xenoantigen, the covalent linkage of the scaffold with the xenoantigen, or a combination thereof. For instance, the crosslinking agent can be derived from N-hydroxysulfosuccinimide.
In certain preferred embodiments, the cross-coupling reaction is carried out in the pH range of 4 to 7.
In certain preferred embodiments, the cross-coupling reaction is carried out in the temperature range of 0xc2x0 C. and 40xc2x0 C.
In certain preferred embodiments, the cross-coupling reaction is carried out a temperature, pH, reactant concentration and for a time sufficient to yield a polymer having a nominal molecular weight of 100,000-500,000 daltons. In certain embodiments, e.g., for tolerogenic forms of the, xenopolymer, the cross-coupling reaction is carried out a temperature, pH, reactant concentration and for a time sufficient to yield a polymer having a nominal molecular weight less than 100,000 daltons.
In certain preferred embodiments, the xenopolymer of the instant invention induces B cell anergy for the xenoantigen.
For many applications, the subject xenopolymer is formulated with a pharmaceutical excipient, and is preferably formulated as a sterile formulation.
Another aspect of the present invention is a method for manufacture of a medicament using the subject xenopolymer, the medicament being administered to a patient which is to receive, or has received, a discordant tissue graft, and in an amount sufficient to reduce the severity of rejection of the graft.
In preferred embodiments, the medicament is formulated with a xenopolymer having a therapeutic index for preventing discordant graft rejection of at least 10.
Another aspect of the present invention provides a kit, including two or more of the subject xenopolymers, each polymer isolated from the other and homogenous with respect to the xenoantigen displayed thereon but different from at least one other polymer of the kit.
Another aspect of the present invention provides a polymer comprising a plurality of subunits interconnected by crosslinking moieties. See generally, FIG. 25. Each crosslinking moiety is covalently bound to at least two subunits, although the individual bonds between a crosslinker and a subunit need not be of the same type or comprise the same atom(s) of the subunits. The individual subunits comprise a backbone or scaffold, and a plurality of xenoantigens. The xenoantigens are covalently tethered to the backbone or scaffold, typically via a tether consisting of between 10 and 20 non-hydrogen atoms. The backbone or scaffold may itself be a polymer, e.g., an oligoethylene glycol, oligosaccharide, or oligopeptide. The synthesis of a polymer of this embodiment of the present invention may be achieved in a single reaction or in a series of reactions. When the synthesis of the polymer occurs in a single reaction, the process comprises combining the backbone or scaffold with the crosslinking agent and the xenoantigens to give a product comprising subunits, i.e., individual backbone or scaffold groups to which xenoantigens have been tethered, that are covalently interconnected via crosslinking moieties. See FIG. 25. When the synthesis of the polymer occurs in a series of reactions, the overall process comprises: combining the backbone or scaffold with the xenoantigens under conditions where the two react to generate subunits bearing a plurality of tethered xenoantigens; and combining the subunits bearing a plurality of tethered xenoantigens with the crosslinking agent to form crosslinks between the individual subunits.
Certain embodiments of this aspect of the present invention provide a polymer, comprising a polymer backbone of polyethylene glycol subunits, at least a portion of which have attached thereto a saccharide moiety represented in the following general formula: 
wherein
R1, independently for each ocurrence, is H, xe2x80x94OR, xe2x80x94SR or xe2x80x94N(H)xe2x80x94Yxe2x80x94R;
R5 is H, xe2x80x94OR, NHxe2x80x94Yxe2x80x94R2, or xe2x80x94Lxe2x80x94E;
Y independently for each occurrence is absent, xe2x80x94C(O)xe2x80x94, xe2x80x94C(S)xe2x80x94, xe2x80x94C(NR)xe2x80x94, O, S, or Se;
B, for one occurrence is xe2x80x94R, and for the other occurrence is xe2x80x94R or from 1-9 saccharide residues;
R, independently for each occurrence, represents H or a C1-C6 alkyl, a protecting group, or xe2x80x94Lxe2x80x94E;
R2 is a C1-C6 alkyl;
X2 represents a linker of 1-10 atoms in the length;
L represents a linker of 1-20 atoms in length, e.g., preferably being a cyclic, branched or straight chain aliphatic group of 2-15 bonds in length, cycloalkyl, alkenyls, cycloalkenyl, alkynyl, aryl, heteroalkyl, or heteroaryl moiety; and
E represents a second PEG subunit of the polymer, wherein, independently for each occurrence of E, the linker group L is covalently attached via R5 or B of a saccharide moiety of the second PEG subunit, or via an amine of the second PEG subunit or via an amide moiety in the tether between the saccharide and the corresponding polyethylene glycol subunit.
Certain embodiments of this aspect of the present invention provide a polymer, comprising a polymer backbone of polyethylene glycol subunits, at least a portion of which have attached thereto a saccharide moiety represented in the general formula (VII): 
wherein,
R1, independently for each ocurrence, is H, xe2x80x94OR, xe2x80x94SR or xe2x80x94N(H)xe2x80x94Yxe2x80x94R;
R5 is H, xe2x80x94OR, NHxe2x80x94Yxe2x80x94R2, or xe2x80x94Lxe2x80x94E;
Y independently for each occurrence is absent, xe2x80x94C(O)xe2x80x94, xe2x80x94C(S)xe2x80x94, xe2x80x94C(NR)xe2x80x94, O, S, or Se;
B, for one occurrence is xe2x80x94R, and for the other occurrence is xe2x80x94R or from 1-9 saccharide residues;
R, independently for each occurrence, represents H or a C1-C6 alkyl or other hydroxyl protecting group;
R2 is a C1-C6 alkyl;
X2 represents a linker of 1-10 atoms in the length;
D represents H, or xe2x80x94Lxe2x80x94E;
L represents a linker of 1-20 atoms in length; and
E represents a second PEG subunit of the polymer, wherein, independently for each occurrence of E, the linker group L is covalently attached via R5 or B of a saccharide moiety of the second PEG subunit, or via an amine of the second PEG subunit.
Still another aspect of the present invention is directed to a polymer, represented by the general formula 
wherein
R1, independently for each ocurrence, is H, xe2x80x94OR, xe2x80x94SR or xe2x80x94N(H)xe2x80x94Yxe2x80x94R;
R5 is H, xe2x80x94OR, NHxe2x80x94Yxe2x80x94R2, or xe2x80x94Lxe2x80x94E;
Y independently for each occurrence is absent, xe2x80x94C(O)xe2x80x94, xe2x80x94C(S)xe2x80x94, xe2x80x94C(NR)xe2x80x94, O, S, or Se;
B, for one occurrence is xe2x80x94R, and for the other occurrence is xe2x80x94R or from 1-9 saccharide residues;
R, independently for each occurrence, represents H or a C1-C6 alkyl or other hydroxyl protecting group;
R2 is a C1-C6 alkyl;
X2 represents a linker of 1-10 atoms in the length
D represents H, or xe2x80x94Lxe2x80x94E;
L represents a linker a linker of 1-20 atoms in length;
E represents another polyethylene glycol subunit of the polymer having a saccharide moiety VII attached thereto, wherein, independently for each occurrence of E, the linker group L is covalently attached via R5 or B in the saccharide moiety.
PEG represents a polyethylene glycol subunit; and
X3 represents one or more additional polyethylene glycol subunit which may be derivatived with a a saccharide moiety of formula VII,
wherein the polymer has an average molecular weight in the range of 10,000 daltons to 1,000,000 daltons.
Still another aspect of the invention relates to a composition including: a non-immunogenic pharmacologically acceptable carrier; and multiple xcex1Gal(1,3)Gal moieties covalently linked to said carrier.
The invention is also directed to a composition useful for reducing plasma levels of anti-(xcex1Gal(1,3)Gal) antibodies in a primate subject in need of a pig organ transplant. Herein, the term xe2x80x9cprimatexe2x80x9d is understood to include humans. Reduction of levels of circulating anti-(xcex1Gal(1,3)Gal) antibodies is expected to prevent or ameliorate hyperacute rejection of the donated pig organ, thereby allowing long-term function of the transplanted organ.
The composition of the invention comprises a non-immunogenic pharmacologically acceptable carrier covalently linked to multiple xcex1Gal(1,3)Gal moieties.
The invention is also directed to methods for synthesizing the composition.
A preferred method is to react in a solution at a pH of 4-7 the following reagents:
a branched polymer, exemplified by polyethylene glycol, which has at least three arms and a minimum of 6 active groups such as amino groups or hydrazino group at its free ends,
xcex1Gal(1,3)Gal having a covalently linked carboxy group, and
N-(3-dimethylaminopropyl)-Nxe2x80x2-ethylcarbodiimide (EDC).
A second preferred method is conducted as above, except the branched polymer has covalently linked carboxy groups, and the xcex1Gal(1,3)Gal has a covalently linked active group such as an amino or hydrazino group.
A third preferred method is to react in a solution at a pH range of 4 to 7 the following reagents:
a branched polymer having at least three arms, and at least six active groups such as amino or hydrazino groups covalently linked to the free ends of the arms, and
xcex1Gal(1,3)Gal having a covalently linked aldehyde.
A fourth preferred method is conducted as the third method above, except that the branched polymer has at least six aldehydes covalently linked to its arms, and the xcex1Gal(1,3)Gal has a covalently linked amino group or hydrazino group.
A fifth preferred method is to react in a solution at a pH range of 4 to 7 the following reagents:
a branched polymer having at least three arms and at least three covalently linked active groups such as amino or hydrazino groups,
xcex1Gal(1,3)Gal having a covalently linked carboxy group,
EDC, and
N-hydroxysulfosuccinimide (NHSS).
A sixth preferred method is conducted as the fifth method above, except that the branched polymer has covalently linked carboxy groups and the xcex1Gal(1,3)Gal has covalently linked amino or hydrazino groups.
The invention also provides methods for reducing plasma levels of anti-(xcex1Gal(1,3)Gal) antibodies in primate subjects, including human subjects, by administering the composition of the invention.
The invention is also directed to a kit and methods for detecting and quantifying anti-(xcex1Gal(1,3)Gal) antibody secreting cells in the blood of a subject.
Yet another aspect of the invention provides a method for attenuating rejection of tissues from a donor animal of one species transplanted to a recipient animal of another species, comprising administering to the recipient animal an amount of a polymer reactive with xenoreactive antibodies of the recipient animal effective for delaying or lessening the severity of a graft rejection response, wherein the polymer has epitopes cross-reactive with an xenoantigen of the graft, and rejection of the tissue is mediated at least in part by the expression of the xenoantigen on the tissue.
The subject method can be used as part of a pre-treatment program for a patient who is to be transplanted with tissue or cells from a discordant species, and/or for treatment of a patient who has been transplanted with tissue or cells from a discordant species. In preferred embodiments, the method reduces or otherwise attenuates the severity of hyperacute rejection of the transplanted tissues. In certain embodiments, the polymer is administered in an amount sufficient to neutralize host antibodies immunoreactive with the xenoantigen.
In preferred embodiments, the receipient does not possess an endogenous UDP galactose:xcex2-D-galactosyl-1,4-N-acetyl-D-glucosminide xcex1(1,3) galactosyltransferase (xcex11,3-GT) activity or does not otherwise produce or display the xcex1Gal(1,3)Gal xenoantigen on its cells, tissues or organs. For instance, the recipient can be a human or old world primate.
In certain preferred embodiments, the xenograft is of pig origin, more preferably minature swine.
The xenotransplanted tissue can be an organ, e.g., a vascularized organ, such as a kidney, heart, lung or liver. The xenotransplant tissue may also be in the form of parts of organs, cell clusters and glands, such as pancreatic islet cells, skin, corneal tissue and bone marrow or other preparations of hematopoietic cells.
In one embodiment, there is provided a method for reducing plasma levels of anti-(xcex1Gal(1,3)Gal) antibodies in a primate subject, said method comprising administering to said subject a composition comprising:
a non-immunogenic pharmacologically acceptable carrier; and
multiple xcex1Gal(1,3)Gal moieties covalently linked to said carrier.
Still another aspect of the present invention provides a complex comprising one or more B-cells associated with a composition comprising:
a non-immunogenic pharmacologically acceptable carrier; and
multiple xcex1Gal(1,3)Gal moieties covalently linked to the carrier.
Yet another aspect of the present invention provides a complex comprising one or more antibodies associated with a composition comprising:
a non-immunogenic pharmacologically acceptable carrier; and
multiple xcex1Gal(1,3)Gal moieties covalently linked to said carrier.
Another feature of the instant invention provides a kit for detecting and quantifying anti-(xcex1Gal(1,3)Gal) antibody-secreting cells in the blood of a subject comprising:
a multi-well plate to which xcex1Gal(1,3)Gal is covalently coupled;
blocking solution comprising a buffer and human serum albumin;
serum-free complete culture medium;
anti-human immunoglobulin antibodies; and
means for labeling said anti-human immunoglobulin antibodies bound to the plate.
Still another feature to the instant invention is a method for detecting and quantifying anti-(xcex1Gal(1,3)Gal) antibody-secreting cells in the blood of a subject comprising:
providing peripheral blood mononuclear cells isolated from a blood sample of said subject;
providing a multi-well plate to which xcex1Gal(1,3)Gal is covalently coupled;
incubating said peripheral blood mononuclear cells on said plate for a sufficient time and under appropriate conditions to allow
anti-(xcex1Gal(1,3)Gal) antibody-secreting cells and their antibodies to bind specifically to said xcex1Gal(1,3)Gal coated on said plate;
washing said plate to remove non-binding cells;
incubating said anti-(xcex1Gal(1,3)Gal) antibody-secreting cells and their antibodies bound to said xcex1Gal(1,3)Gal coated on said plate with anti-human immunoglobulin antibodies;
washing said plate to remove non-binding anti-human immunoglobulin antibodies;
applying a means to label anti-human immunoglobulin antibodies bound to said plate; and,
quantifying single anti-(xcex1Gal(1,3)Gal) antibody-secreting, spot forming cells indicated by spots formed on said plate by the binding of said labelled anti-human immunoglobulin antibodies.